Regenerative effects of
mesenchymal stem cell-derived exosomes
Xiaoqin Wang 王晓勤
Department of Biomaterials Institute of Clinical Sciences
Sahlgrenska Academy, University of Gothenburg
Gothenburg 2019
Cover illustration: Left: MSC adherent on exosome-immobilised titanium surface, image by Furqan Ali Shah. Right: MSC internalised PKH67-labelled exosomes (green), image by Xiaoqin Wang.
Regenerative effects of mesenchymal stem cell-derived exosomes
© Xiaoqin Wang 2019
xiaoqin.wang@biomaterials.gu.se ISBN 978-91-7833-340-0 (PRINT) ISBN 978-91-7833-341-7 (PDF) Printed in Gothenburg, Sweden 2019 Printed by BrandFactory
To my beloved family
不忘初心， 方得始终
stem cell-derived exosomes
Xiaoqin Wang
Department of Biomaterials, Institute of Clinical Sciences Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
ABSTRACT
Mesenchymal stem cells (MSCs) play pivotal roles for bone regeneration by virtue of their osteogenic differentiation ability and immunomodulatory capacity. Recently, secretion of exosomes/extracellular vesicles (EVs) has been suggested as a new mechanism of MSC-based therapy. MSC-derived EVs/exosomes have shown promising effects in tissue regeneration and immunomodulation, which are attributed to their regulatory effects in various processes. The overall objective of this thesis was to explore the cell-to-cell communication and cell-to-material surface interaction mediated by MSC- derived EVs/exosomes. The emphasis was placed on their functions in the regeneration capacity of MSCs and the determination of the microRNA and protein contents of these EVs/exosomes in order to obtain an insight into the underlying mechanisms of the EV-/exosome-mediated biological effects.
The results demonstrated that exosomes secreted from MSCs in the mid and late stage of osteogenic differentiation induced osteogenic lineage commitment, but only exosomes from the late differentiation induced the mineralisation of the extracellular matrix. MSC-derived exosomes were internalised by a subpopulation of homotypic cells. The differentially expressed microRNAs were osteogenesis related and predicted to enrich pathways involved in the regulation of osteogenic differentiation and general mechanisms by which exosomes exert their functions. In vitro ageing increased the secretion of EVs and in some contexts altered the protein profiles of EVs.
The top abundant proteins in high passage (HP, “aged”) and low passage (LP,
“young”) EVs shared similar but not identical functional features with an overlap of the enriched pathways related to endocytosis and regulation of cell proliferation and survival. The differentially expressed proteins in HP EVs were predicted to enrich GO biological process related to transport and secretion. Both HP and LP EVs promoted MSC proliferation in autocrine and paracrine manners and in a dose-dependent fashion. In contrast to MSC-
titanium (Ti) surfaces accelerated and increased MSC adhesion, influenced the early morphology and promoted the growth of MSCs on titanium. Proteomic analysis of the exosomal protein revealed identified proteins with predicted GO molecular function related to adhesion, structure and morphology, and growth factor and growth factor receptor activity.
In conclusion, MSC-derived EVs/exosomes possess regenerative effects, in terms of the stimulation of the proliferation and osteogenic differentiation of MSCs, and influence the behaviour of MSCs on titanium surfaces. The expression of exosomal cargoes is altered during osteogenic differentiation and in vitro ageing and their predicted functions partially correspond to the observed effects. It is suggested that the MSC-derived EV-/exosome-mediated effects on the regeneration capacity of MSCs are at least partially attributed to the transfer of functional exosomal cargoes.
Keywords: aging, cell adhesion, cell-material interaction, exosomes, extracellular vesicles, mesenchymal stem cells, osteogenic differentiation, proliferation, regeneration, titanium
ISBN 978-91-7833-340-0 (PRINT) ISBN 978-91-7833-341-7 (PDF)
Mesenkymala stamceller (MSC) är en typ av adulta stamceller som är viktiga för benregeneration. MSC kan både differentiera till benceller samt påverka andra celler immunologiskt och på så vis påverka benregeneration. Exosomer är en typ av extracellulära vesiklar (EV) som frisätts från celler till den extracellulära miljön och som kan fungera som budbärare mellan celler. De kan interagera med celler i närmiljön eller transporteras i blodet till mer distala celler. EV/exosomer frisätts från de flesta, om inte alla, celler och deras innehåll och funktion korrelerar med vilken celltyp och under vilket stimuli/miljö de frisätts. MSC exosomer har i olika studier bland annat visats påverka olika regenerationsprocesser som benläkning, hjärtregeneration och njurregeneration.
Syftet med den här avhandlingen var att utforska EV/exosomer från MSC med fokus på deras funktion vid regeneration samt deras protein- och mikroRNA- innehåll. Vidare avsåg vi att studera deras roll vid cell-cell kommunikation samt cell-material interaktion. Vi har undersökt detta närmare i tre olika studier. I den första studien studerade vi exosomer/EV som frisatts från MSC när dessa differentieras till osteoblaster. Vi undersökte exosomer/EV närmare vid tre olika stadier; expansion, tidig och sen differentiering. Studien visade att exosomer/EV frisätts under hela differenteringsprocessen. EV/exosomer från differentierade celler kan påverka andra MSC att börja differentiera till osteoblaster men endast när exosomer/EV kommer från sent differentierade celler kan de påverka andra celler att mineralisera den extracellulära matrisen.
Vi kunde också identifiera osteogenrelaterade mikroRNA i EV/exosomer som åtminstone delvis kan förklara effekten inducerad av EV/exosomer.
I den andra studien undersökte vi hur MSC frisätter EV/exosomer när de åldras och deras funktion på både ”unga” och ”gamla” MSC. För att undersöka detta använde vi oss av en in vitro modell där MSC odlades från låga passager (LP,
”unga” celler) och höga passager (HP, ”gamla” celler). Vi kunde visa att
”gamla”, HP MSC frisätter mer EV/exosomer jämfört med ”unga”, LP MSC.
Båda typerna av vesiklar kunde öka proliferation och överlevnad av både
”unga” och gamla” celler. Vidare så kunde vi detektera nästan 2000 olika proteiner i de olika typerna av EV, varav många hade med cellproliferation och överlevnad att göra.
hur exosomer fästa till ytor påverkar MSC med avseende på celladhesion, proliferation och differentiering. Vidare jämförde vi det med EV/exosomer som tillsatts direkt till cellodlingsmediet eller med ytor utan några EV/exosomer. För detta använde vi oss av två grupper av exosomer/EV;
isolerade från MSC som prolifererar samt från MSC som differentierar. Vi kunde visa att titanytor med ytbundna exosomer stimulerade adhesionen av MSC, påverkade cellernas morfologi och ökade cellernas proliferation. Med hjälp av proteomik kunde proteiner detekteras i exosomerna som är av betydelse för celladhesion, morfologi och tillväxt.
Sammanfattningsvis har denna avhandling visat att MSC bildar och frisätter EV/exosomer med regenerativa effekter vad avser stimulering av proliferation och osteogen differentiering av MSC samt påverkan av MSC på titanytor.
Exosomernas innehåll förändras under osteogen differentiering och åldrande in vitro och deras förutsagda funktioner motsvarar delvis de observerade cellulära effekterna.
This thesis is based on the following studies, referred to in the text by their Roman numerals.
I. Wang X, Omar O, Vazirisani F, Thomsen P, Ekström K.
Mesenchymal stem cell-derived exosomes have altered microRNA profiles and induce osteogenic differentiation depending on the stage of differentiation.
PLoS One. 2018; 13(2): e0193059.
II. Wang X, Philip J, Vazirisani F, Tsirigoti C, Thomsen P, Ekström K.
The impact of in vitro aging on the release of extracellular vesicles from human mesenchymal stem cells.
In manuscript.
III. Wang X, Shah FA, Vazirisani F, Johansson A, Palmquist A, Omar O, Ekström K, Thomsen P.
Exosomes influence the behavior of human mesenchymal stem cells on titanium surfaces.
Submitted for publication.
List of papers not included in the thesis
I. Ekström K, Omar O, Granéli C, Wang X, Vazirisani F, Thomsen P.
Monocyte exosomes stimulate the osteogenic gene expression of mesenchymal stem cells.
PLoS One. 2013;8(9): e75227.
II. Nawaz M, Camussi G, Valadi H, Nazarenko I, Ekström K, Wang X, Principe S, Shah N, Ashraf NM, Fatima F, Neder L, Kislinger T.
The emerging role of extracellular vesicles as biomarkers for urogenital cancers.
Nat Rev Urol. 2014;11(12): 688 - 701. Review.
ABBREVIATIONS ... VI
1 INTRODUCTION ... 1
1.1 Extracellular vesicles/exosomes ... 1
1.1.1 Classification and nomenclature ... 1
1.1.2 Biogenesis of EVs/exosomes ... 3
1.1.3 Molecular composition of EVs/exosomes ... 6
1.1.4 Isolation of EVs/exosomes ... 9
1.1.5 Characterisation of EVs/exosomes ... 10
1.1.6 EV-/exosome-mediated cell-to-cell communication ... 12
1.2 Mesenchymal stem cells (MSCs) ... 14
1.2.1 Historical background of MSCs ... 14
1.2.2 Characterisation and sources of MSCs ... 15
1.2.3 Osteogenic differentiation of MSCs ... 16
1.2.4 Immunoregulatory effects of MSCs ... 19
1.2.5 Ageing-related changes of MSCs ... 20
1.2.6 Secretome of MSCs ... 21
1.3 MSC-derived EVs/exosomes ... 23
1.3.1 Cargoes of MSC-derived EVs/exosomes ... 23
1.3.2 Functions of MSC-derived EVs/exosomes ... 29
1.4 Bone formation around implants ... 36
1.4.1 Cell-material surface interactions ... 37
1.4.2 Titanium surface modification ... 38
2 AIM ... 41
2.1 Specific aims ... 41
3 MATERIALS AND METHODS ... 43
3.1 Materials ... 43
3.1.1 MSCs ... 43
3.1.2 Titanium discs ... 43
3.2.1 Expansion and in vitro ageing of MSCs ... 43
3.2.2 Osteogenic differentiation of MSCs ... 44
3.2.3 Conditioned media collection ... 44
3.2.4 Characterisation of MSCs ... 44
3.3 EV/exosome isolation ... 45
3.3.1 Ultracentrifugation ... 45
3.3.2 Exo-spin isolation ... 45
3.4 EV/exosome characterization ... 46
3.4.1 Nanoparticle tracking analysis (NTA) ... 46
3.4.2 Western blot ... 46
3.4.3 Transmission electron microscopy (TEM) ... 47
3.5 Delivery of EVs/exosomes ... 47
3.5.1 Delivery via suspension ... 47
3.5.2 Delivery via immobilisation on titanium surfaces ... 48
3.6 EV/exosome labelling and uptake ... 48
3.7 Staining of MSCs ... 48
3.7.1 Alizarin red staining ... 48
3.7.2 Actin red staining ... 49
3.8 Microscopy and image analysis ... 49
3.8.1 Wide field fluorescence microscopy and confocal microscopy .. 49
3.8.2 Scanning electron microscopy (SEM) ... 49
3.8.3 Image analysis ... 50
3.9 Colorimetric assays ... 50
3.9.1 microBCA assay ... 50
3.9.2 Cell adhesion and growth assay ... 50
3.9.3 Lactate dehydrogenase assay ... 51
3.9.4 Alkaline phosphatase activity ... 51
3.9.5 Extracellular matrix mineralisation ... 51
3.10Gene expression analysis ... 52
3.10.2Quantitative polymerase chain reaction (qPCR) ... 52
3.10.3MicroRNA profiling ... 52
3.11Mass spectrometry of proteomic analysis ... 53
3.12Bioinformatic analyses ... 54
3.12.1MicroRNA target prediction and pathway analysis ... 54
3.12.2Functional prediction of identified proteins ... 54
3.13Statistical analyses ... 55
4 SUMMARY OF RESULTS ... 57
4.1 Paper I ... 57
4.2 Paper II ... 59
4.3 Paper III ... 63
5 DISCUSSION ... 67
5.1 Methodological consideration ... 67
5.1.1 Cell types, sources and culture conditions ... 67
5.1.2 Purity of MSC-derived EVs/exosomes ... 71
5.1.3 Normalisation of MSC-derived EVs/exosomes ... 72
5.2 Contents of MSC-derived EVs/exosomes ... 73
5.2.1 microRNAs ... 73
5.2.2 Proteins ... 75
5.3 Effects of MSC-derived EVs/exosomes ... 78
5.3.1 Effects on osteogenic differentiation ... 78
5.3.2 Effects on proliferation ... 80
5.4 The behaviour of MSCs on exosome-immobilised titanium surfaces . 81 5.5 Potential application of MSC-derived EVs/exosomes ... 86
6 SUMMARY AND CONCLUSION ... 89
7 FUTURE PERSPECTIVES ... 91
ACKNOWLEDGEMENT ... 93
REFERENCES ... 97
Ab b-amyloid
AD Alzheimer disease
AFM atomic force microscopy
Ago2 argonaute-2
AIH autoimmune hepatitis
AKI acute kidney injury
ALIX apoptosis-linked gene 2-interacting protein X
ALP alkaline phosphatase
ALT alanine transaminase
AR androgen receptor
ARF6 ADP ribosylation factor 6
AST aspartate aminotransferase
AT-MSC adipose tissue-derived MSCs
ATP adenosine triphosphate
b-TCP tricalcium phosphate
BCA bicinchoninic acid
(b)FGF (basic) fibroblast growth factor
BGM basal growth media
BM-MSC bone marrow derived MSC
BMP-2 bone morphogenetic protein-2
c-JNK c-Jun N-terminal kinases
CCI4 carbon tetrachloride
CCK-8 Cell counting kit-8
CDC42 cell division cycle 42
CM conditioned media
CP-MSC chorionic plate-derived MSC
cryo-EM cryo-electron microscopy
CTGF connective tissue growth factor
CXCR4 C-X-C chemokine receptor type 4
DAPI 4′,6-diamidino-2-phenylindole
DC dendritic cell
DLL4 delta-like 4
DLS dynamic light scattering
DMEM-LG Dulbecco’s modified Eagle’s medium with low glucose
ECM extracellular matrix
EFNA3 Ephrin A3
EGFR epidermal growth factor receptor
EMT epithelia-to-mesenchymal transition
ER endoplasmic reticulum
ESCRT endosomal sorting complex required for transport
EV extracellular vesicle
FBS fetal bovine serum
FZD Frizzled
GA glioma-associated
GF growth factor
GFR growth factor receptor
GO gene ontology
Grp 94 glucose-regulated protein 94
GSC glioma stem-like cell
GSH glutathione
GSK glycogen synthase kinase
GVHD graft-versus-host disease
(h)FN (human) fibronectin
HGF hepatocyte growth factor
Hh hedgehog
HIF-1a hypoxia inducible factor-1a
HP high passage
hPBMC human peripheral blood mononuclear cell
HRS/HGS hepatocyte growth factor-regulated tyrosine kinase substrate
HSC hematopoietic stem cells
HSC70 (Hsp70) heat shock protein 70
HUVEC human umbilical vein endothelial cell
ICAM intracellular adhesion molecule
IFNg interferon gamma
IGF/IGFR insulin-like growth factor/insulin-like growth factor receptor
IL/ILR interleukin/interleukin receptor
ILV intraluminal vesicles
IPA Ingenuity pathway analysis
IRAK1 interleukin 1 receptor associated kinase 1
LDH lactate dehydrogenase
LP low passage
LRP low-density lipoprotein receptor-related protein
LUAD lung adenocarcinoma
M-PER mammalian protein extraction reagent
MAPK mitogen-activated protein kinase
MCAo middle cerebral artery occlusion MCP-1 monocyte chemoattractant protein-1
MDA malondialdehyde
MFG-E8 milk fat globule epidermal growth factor 8
MHC major histocompatibility complex
MI myocardial infarction
MPO myeloperoxidase
MSC mesenchymal stem cell
mTOR mammalian target of rapamycin
MVB multivesicular body
Myo1e myosin 1E
NCOR1 nuclear receptor co-repressor 1
NEP neprilysin
NF-kB nuclear factor-kB
nLC-MS nano-liquid chromatography coupled to an Orbitrap mass spectrometer
NLRP3 NACHT, LRR and PYD domains containing protein 3
nSMase neutral sphingomyelinase
NTA nanoparticle tracking analysis
OCPC ortho-cresolphthalein complexone
ODM osteogenic differentiation media
OPG osteoprotegerin
PBS phosphate buffer saline
PD(T) population doubling (time)
PDGF platelet-derived growth factor
PGE2 prostaglandin E2
PI3K/Akt phosphatidylinositol 3-kinase/protein kinase B
PLD2 phospholipase D2
PM plasma membrane
PS phosphatidylserine
PTEN phosphatase and tensin homolog
qPCR quantitative polymerase chain reaction Rab (RAB) Ras-related proteins in brain
RhoA ras homolog gene family member A
RNPs ribonucleoproteins
RT room temperature
RUNX2 runt-related transcription factor 2
SASP senescence-associated secretory phenotype SDF-1α stromal cell-derived factor 1α
SEC size exclusion chromatography
SEM scanning electron microscopy; standard error of mean
SM sphingomyelin
SOD superoxide dismutase
STAB2 stabilin 2
STAM signal transducing adaptor molecule
STAT3 signal transducer and activator of transcription 3
Stau staufen
TCPS tissue culture treated polystyrene
TEM transmission electron microscopy
TGFb transforming growth factor beta
Ti titanium
TLR toll-like receptor
TMT tandem mass tag
TNF-a tumour necrosis factor-a
tRNA transfer RNA
TRPS tunable resistive pulse sensing
Tsg101 tumor susceptibility gene 101 protein
UC umbilical cord
VEGF(R) vascular endothelial growth factor (receptor) VPS4 vacuolar protein sorting-associated protein 4
VSMC vascular smooth-muscle cell
1 Introduction
1.1 Extracellular vesicles/exosomes
Extracellular vesicles (EVs) are particles that are naturally released from the cell and delimited by a lipid bilayer, containing cytosol from the secreting cells, but not containing a functional nucleus, indicating the lack of ability to replicate [1]. The secretion of EVs appears to be a conserved process throughout evolution, as both eukaryotic and prokaryotic cells have been reported to release vesicles into the extracellular space. However, in the present thesis, the term “EVs” only refers to vesicles secreted by eukaryotic cells. An early evidence of the presence of EVs was reported in 1969, in which membrane-enclosed vesicles, named as “matrix vesicles”, were found to locate in the matrix of cartilage and to be associated with calcification [2]. For decades, EVs were found in different biological fluids and secreted by various of mammalian cells. Nevertheless, these vesicles were initially assumed to be secreted by the outward budding of the plasma membrane (PM) of cells. In 1983, another more complicated EV secretion pathway was demonstrated by Harding et al. [3] and Pan and Johnstone [4], in which vesicles were formed in the intracellularly endosome pathway, particularly in multivesicular bodies (MVBs), and secreted by the fusion of MVBs with the PM, resulting in the release of intraluminal vesicles (ILV) into the extracellular space. Thereafter, the term “exosome” was proposed by Johnstone et al. in 1987 to describe the released vesicles of endosomal origin [5]. However, for a long time, exosomes were assumed to function as a “waste bin” for cells to dispose of unwanted components. A great deal of attention was then paid to another breakthrough in 2007, showing that exosomes, containing mRNA and microRNA, mediated cell-to-cell communication via the transfer of genetic material [6]. As of today, EVs/exosomes research is growing exponentially and a huge number of studies have been published.
1.1.1 Classification and nomenclature
EVs are heterogeneous because of their diverse origin, nature and features.
Over the years, EVs have been classified in different ways and assigned various names based on size, biogenesis, cell origin or function. Currently, a commonly accepted way to classify EVs is based on their biogenesis, in which EVs are categorised into three broad classes: exosomes, microvesicles/
ectosomes and apoptotic bodies [7]. Apoptotic bodies are vesicles formed during apoptosis when the cell cytoskeleton breaks and the PM bulges outwards. Apoptotic bodies contain parts of a dying cell and consist of vesicles with the most heterogeneous diameter size ranging from 200 nm to 5 µm [7, 8]. Microvesicles/ectosomes are thought to be formed by the outward budding of the PM of viable cells, in a size range of 100 nm to 800 nm [9]. Exosomes are of endosomal origin and are thought to have the smallest size range (30 nm- 150 nm) among all three groups of EVs. However, different classifications have been proposed as a result of the knowledge that has been accumulated on the diversity of EVs. This is illustrated by the overlap of size and density among different subpopulations of EVs and the much more diverse morphology of EVs than previously observed [10, 11], as well as the challenge encountered in isolating a pure subpopulation of EVs based on the currently available techniques.
Along with the development of the EV research field, more and more evidence shows the overlaps between different subgroups of EVs. A generic means of classification and nomenclature has therefore recently been suggested [12].
EVs can be classified based on their physical characteristics, such as size, density and morphology, or based on their biochemical composition, for example, positive for a specific molecule, or referring to the secretion conditions or cell origin. The following are two examples using the recently proposed generic means of classification and nomenclature [13]. When applying size as the criterion for classification, the use of the generic terms small EVs and middle/large EVs for vesicles of < 200 nm and > 200 nm respectively is suggested. Another alternative way is to classify EVs based on their density, in which vesicles are grouped into low-, middle- and high- density EVs, with each density range defined.
The development of EV research has highlighted some limitations of the current classification and nomenclature and has revealed the need for a consistent classification and nomenclature for exchanging information within the field and communicating with other research fields. Debates and discussions on these issues, i.e., nomenclature, are on-going and efforts to provide new and better technical solutions are emerging. Although different operational terms for EVs have been suggested, “exosome” is still one of the most commonly used terms in the current literature. However, due to the difficulty involved in obtaining solid evidence of the endosomal origin of studied vesicles in reality, the term “exosome” is often used in a less strict
manner compared with the initial definition. Indeed, some researchers classified the subpopulation of microvesicles, which is indistinguishable from exosomes, i.e., sharing a similar size and density, enriching classical markers such as CD63 and CD81, and the formation involved the tumour susceptibility gene 101 protein (TSG101) and the vacuolar protein sorting-associated protein 4 (VPS4), components of the endosomal sorting complex required for transport (ESCRT) machinery, as exosomes [14, 15]. In the present thesis, the term “EVs/exosomes” is used to refer to an exosome-enriched vesicle preparation, isolated using high-speed ultracentrifugation or a commercial chromatography isolation kit, Exospin, and characterised by applying complementary techniques.
1.1.2 Biogenesis of EVs/exosomes
The heterogeneity of EVs reflects the complexity of EVs biogenesis and the existence of various mechanisms and pathways regulating the formation and release of EVs. As previously mentioned, the theory of the direct outward budding of the PM of cells as the main mode for EVs secretion was replaced by the finding that the endosomal pathway was demonstrated for the formation of MVB, in which ILVs were formed by the inward budding of the endosomal membrane and subsequently exocytosed into the extracellular environment as exosomes [2, 4, 5, 16]. For a long time, EVs budded directly from the PM and formed via the endosomal pathway were considered to be regulated by distinguishable machineries. However, growing evidence shows that the same machineries can play similar roles either at the PM for direct budding or in the intracellular endosomal compartments for the biogenesis of exosomes [15, 17- 21]. These observations indicate that similar yet not identical machineries exist to regulate the biogenesis of different subpopulations of EVs/exosomes.
Although the biogenesis of EVs and involved mechanisms are as yet incompletely understood, current knowledge has implicated ESCRT- dependent and ESCRT-independent mechanisms (Figure 1). Upon endocytosis, early endosomes, with tubular extensions, form intracellularly.
Following maturation, ILVs are assembled by the inward budding of late endosomal membrane to form MVB. The most described mechanism in driving the formation of ILVs and MVB is ESCRT-dependent machinery. The human ESCRT consists of 33 proteins which are assembled into four complexes, ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III, with associated proteins VPS4 complex and Bro1 domain proteins including apoptosis-linked
gene 2-interacting protein X (ALIX) [22]. The four complexes are numbered according to the order in which they act in the pathway and play distinct roles.
ESCRT-0, consisting of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS, also known as HGS) and signal transducing adaptor molecule (STAM), together with clathrin, recognise and sequester ubiquitinated transmembrane proteins in the endosomal membrane. Following the recruitment of TSG101 of the ESCRT-I complex by HRS, the ESCRT-II complex is recruited via ESCRT-I and together initiate the local budding of the endosomal membrane with sorted cargo. ESCRT-III participates in protein deubiquitination and subsequently drives vesicle scission [1, 9, 22].
Thereafter, MVBs, loading with ILVs, are formed and undergo either fusion with lysosomes, resulting in the discharge and digestion of their ILVs in the lumen of lysosomes, or fusion with the PM, leading to the release of exosomes into the extracellular space.
Figure 1. Biogenesis of EVs/exosomes. The biogenesis of exosomes and PM- derived EVs is regulated by both ESCRT-dependent and ESCRT-independent mechanisms. (Figure inspired from [1])
An increasing number of studies have provided a comprehensive overview on the role of individual ESCRT proteins in exosome biogenesis and secretion.
RNA interference in HeLa cells to silence ESCRT-0 genes, HGS and STAM1,
Lipids Tetraspanins
ESCRTESCRTLipids
Tetraspanins ESCRT-dependent
ESCRT-independent Golgi
complex Early endosome
Lysosome
inward MVB budding
ILV ESCRT PM (Tsg 101, VPS4) ARF6, SMase
or ESCRT-I gene TSG101 decreases exosome secretion [18]. Partially in line with this result, silencing of TSG101in MCF-7 breast tumour cells showed to decrease exosomes secretion [23]. However, contradictory results have been reported when silencing other ESCRT genes in different cell types. Baietti et al. showed that the depletion of ALIX or VPS4B impaired exosome secretion in MCF-7 cells [23], whereas Colombo et al. reported that the exosome secretion was increased by the depletion of ALIX or VPS4B in HeLa cells [18]. Together, these findings indicate the complexity and heterogeneity of mechanisms regulating EV/exosome secretion, which may also be partially contributed by the differences of parental cell types. Moreover, the study demonstrated that the ESCRT machinery not only regulates the amount of exosomes released but is also involved in the exosomal cargo loading [18, 23].
This is supported by the observation of reduced amounts of CD63 and major histocompatibility complex (MHC) class II in exosomes secreted by TSG101 or STAM1 knockdown HeLa cells [18], as well as reduced CD63 in exosomes secreted by TSG101 or ALIX knockdown MCF7 cells [23]. In addition to the regulation of exosome formation through the endocytotic pathway, ESCRT machinery, particularly the TSG101 and VPS4 components, was also involved in the formation of EVs directly budding from the PM. The interaction of TSG101 with a specific (protein) sequence, the PSAP sequence present in the gag protein of retroviruses [15] or arrestin domain-containing protein 1 [24], induced the budding of EVs at the PM.
In addition, ESCRT-independent machinery mediated by lipids, tetraspanins and small GTPase has been implicated in the regulation of EV/exosome biogenesis. The lipid metabolism enzyme, neutral sphingomyelinase (nSMase), has been shown to hydrolyse sphingomyelin (SM) into ceramide, which was required for the transfer of exosome-associated domains into the lumen of endosomes [25]. The inhibitor of nSMase, GW4869, reduced the secretion of proteolipid protein-bearing exosome [25] and the exosomal protein, flotillin 1 [26], as well as exosomal microRNA [27]. Similarly, phosphatidic acid, hydrolysed by phospholipase D2 (PLD2), was also shown to play a role in inward budding and increase of exosome secretion [19, 28]. Various members of tetraspanins, CD63, CD9 and CD82, have also been shown to regulate exosomes formation and secretion [26, 29]. The overexpression of CD9 or CD82 induces the secretion of b-catenin-bearing exosomes [26]. Some evidence also suggests the roles of small GTPase Ras-related proteins in brain (Rab) proteins, RAB11, RAB35, RAB7 and RAB27A/B, in endosome maturation and exosome secretion (reviewed by Stenmark) [30]. In details,
RAB11 and RAB35 were mainly associated with recycling and early endosomes respectively, whereas RAB7 and RAB27 were associated with late endosomal and secretory compartments. In comparison to exosome biogenesis, PM-derived EVs were also shown to be regulated by SMase [17] and small GTPase ADP ribosylation factor 6 (ARF6) [20]. The overexpression of ARF6 depolymerised the actin cytoskeleton and permitted the efficient release of PM-derived EVs. Interestingly, ARF6, together with its effector, PLD2, affected the budding of ILVs into MVBs, suggesting that ARF6 is also involved in the regulation of exosomes formation [19].
1.1.3 Molecular composition of EVs/exosomes
The molecular composition of EVs/exosomes has been studied using different approaches. Comprehensive data have been collected in database such as EVpedia (http://evpedia.info) and Vesiclepedia (http://microvesicles.org/).
The contents of EVs/exosomes often reflect their parental cell sources and are influenced by their secretion conditions. In general, EVs/exosomes contain lipids, proteins and nucleic acids (Figure 2).
Sphingomyelin
Cholesterol
Ceramide (e.g. GTPase Rabs)
Figure 2. Molecular composition of EVs/exosomes. EVs/exosomes enclosed by lipid bilayer contain lipids, proteins and nucleic acids. The main specifics/families of lipids, proteins and nucleic acids that have been detected in EVs/exosomes are presented in the figure. (Figure adapted from [1])
The lipid composition of EVs has not been studied as much as the protein contents. Nevertheless, it has been reported that, in comparison with the total cell membrane, EVs/exosomes are enriched with several lipid species, including cholesterol, SM, phosphatidylserine (PS) and saturated fatty acids [18, 31]. Moreover, a recent study exploring the lipidomes of exosomes derived from three different cell types, Huh7 hepatocellular carcinoma cells, U87 glioblastoma cells and mesenchymal stem cells (MSCs), revealed a similarity in the lipidomes between Huh7 and MSC exosomes, but not U87 exosome [32]. A comparison of the lipidomes of two subpopulations of EVs showed the enrichment of different lipid species [32]. These observations hint at a mechanism that sorts selected lipid species into vesicles, which may be dependent on both cell origin and vesicle types. Such hypothesis is also supported by significantly different levels of specific lipids in urinary exosomes from prostate cancer patients and healthy donors [33]. On the basis of the features of the enriched lipids, it was proposed that the lipid composition contributes to the stability and structural rigidity of vesicles [31]. On the other hand, some studies have demonstrated that the lipids and lipid metabolism play important roles in the regulation of the biogenesis of both endosomal-origin exosomes and PM-derived EVs [17, 19, 25, 28, 34].
The protein contents of EVs have been studied using the antibody-based detection of specific proteins initially [3], while the high-throughput proteomic technique has enabled the large-scale identification of the global proteome of EVs. Early proteomic studies showed that both cell type-dependent and - independent proteins were detected in exosomes. The cell type-independent proteins often came from specific subcellular compartments, including the PM, cytosol and endosomes, and rarely from the nucleus, mitochondria, endoplasmic reticulum (ER) and Golgi apparatus [35, 36]. These proteins were therefore defined as common markers for exosomes, whereas proteins from compartments such as the ER often served as negative markers for exosomes.
The typical enriched exosomal proteins, irrespective of cell origin, include transmembrane proteins such as (i) tetraspanins CD63, CD9, CD81 and CD82, (ii) integrins, (iii) proteins binding to the lipid raft such as flotillin and annexins; cytoskeleton proteins such as actin; the protein components of ESCRT machinery, i.e. TSG101 and ALIX; cytosolic proteins such as heat
shock 70-kDa protein (HSC70) and proteasome; and GTPase Rabs (reviewed in [1, 37]). However, as the cargo-sorting mechanism for different subpopulations of EVs is not yet fully understood, it is not clear whether these enriched exosomal proteins, often serving as markers, are specifically detected in exosomes or whether they are also present in another subpopulation of EVs, i.e. PM-derived EVs. Theoretically, proteins located in the cell membrane or cytosol may also have the chance to be sorted into PM-derived EVs. Indeed, a recent study showed that classical markers for exosomes, such as MHC, flotillin and HSC70, were similarly present in all the studied EVs, indicating the non-specificity of these proteins among subtypes of EVs [38]. Moreover, through a comprehensive comparison of the proteome of EVs recovered by different centrifugation speeds, density gradients and antitetraspanin-coated beads, the authors proposed syntein-1, TSG101 and tetraspanins CD63, CD81 and CD9 as a new panel of specific markers for small EVs, including exosomes [38]. In addition to the cell type-independent proteins, efforts have also been made to compare the protein contents of EVs secreted by different cell types [32, 38] or under various conditions, i.e. pathological or healthy conditions [39, 40], to unravel the specific proteins enriched in EVs that are reflective of cell origin. Recently, a new technique, the multiplex proximity extension assay, has been reported to be able to identify the cellular origin of exosomes recovered from different cell lines and body fluids [41]. Taken as a whole, the development of knowledge and techniques will enable us to acquire a better understanding of the EV biology and application of EVs, i.e. as biomarkers of disease.
Since the first evidence showing that exosomes contain RNA, particularly microRNA and mRNA [6], the RNA contents of EVs have been intensively studied. In addition to microRNA, several other species of non-coding RNAs have been identified in EVs/exosomes, including vault-RNA, Y-RNA and specific transfer RNA (tRNA) [42]. However, it was confirmed that ribosomal RNA was almost absent in EVs/exosomes [43]. The detected RNAs were shown to be resistant to RNase digestion, indicating their intravesical location [6]. Among these detected RNAs, microRNA has been attracted the most attention. A growing body of studies have suggested that microRNA carried via EVs/exosomes as one of the most important populations of extracellular circulating microRNAs, which is involved in the regulation of both physiological and pathological processes [44, 45]. Furthermore, altered microRNA profiles have been found in EVs/exosomes secreted from various differentiation stages of dendritic cells [46] and MSCs [47]. Another study
found that sumoylated hnRNPA2B1 mediated the targeting of microRNA to be sorted in exosomes [48]. These observations suggest the existence of specific sorting mechanisms for RNA cargo in EVs/exosomes, which requires further comprehensive investigation. Interestingly, a few studies have found the presence of DNA, both genomic and mitochondrial DNA, in EVs/exosomes [49-51]. However, a great deal of information is still lacking, i.e. (i) whether DNA is presented in bona fide exosomes or its presence is due to the co-isolation of other subtypes of EVs such as apoptotic bodies that are well-known to contain DNA; (ii) whether DNA is present inside EVs or on the surface of EVs and (iii) whether DNA is specifically sorted inside EVs, like other EVs constituents.
1.1.4 Isolation of EVs/exosomes
EVs/exosomes can be isolated using various techniques, including differential ultracentrifugation, filtration, size exclusion chromatography (SEC), precipitation and immunoaffinity capture (reviewed in [52, 53] ). There is no one-size-fits-all approach. The selection of an isolation method therefore depends on the properties of the starting material (i.e. cell culture media or biological fluid, volume of the material), the aims of the study and the interested downstream analyses.
Differential ultracentrifugation, with or without a further purification step by extra washing or density gradient depending on the requirement of sample purity, is the most commonly used isolation method [54]. On the basis that vesicles with different sizes and density are sedimented by different centrifugation speeds, a low centrifugation speed (i.e. 300xg, 500xg or 2,000xg) is first applied to remove dead cells and cell debris. Following a medium-speed centrifugation (i.e. 10,000xg-20,000xg) to eliminate large vesicles, small vesicles are finally pelleted using high-speed centrifugation (i.e. 100,000xg or 120,000xg) [55]. Traditionally, various subpopulations of vesicles are isolated by different centrifugation steps. Large vesicles like apoptotic bodies are pelleted at 2,000xg or 3,000xg, middle size vesicles, such as PM-derived microvesicles, are isolated at 10,000xg-20,000xg, whereas exosomes are pelleted at 120,000xg [43]. However, it is acknowledged that such separation may not be sufficient, due to the overlapping of vesicle size among different subpopulations of EVs, as well as the existence of vesicles of the same size but different densities [7, 56]. Moreover, non-vesicle structures such as protein aggregates may be co-isolated. One solution partially to solve
these problems is to apply a density gradient. The total EV samples can be sedimented in iodixanol gradients or floated in sucrose gradients and EVs sedimented at specific densities are collected for further study [38]. Similarly, the principle for isolation by filtration or SEC is based on the differences in vesicle size. SEC has been shown efficiently to eliminate soluble proteins, thereby reducing the non-vesicle contamination [57, 58]. Such non-pelleting procedure also avoids the possible damage or aggregate of vesicles, thereby preserving the integrity of vesicles. Commercially, water-excluding polymers such as polyethylene glycol (PEG) have been used for the isolation of EVs [59]. The crude samples are first incubated with precipitation solution and, following a low-speed centrifugation, the precipitate containing vesicles is collected. Isolation by precipitation is simple, with high recovery, but it often results in samples with poor purity. In comparison to the above-described methods, isolation based on immunoaffinity allows to capture vesicles with specific protein markers, such as CD63, CD81 and CD9 [38]. Nevertheless, all the existing methods generally have their pitfalls, necessitating the optimisation of current techniques and the development of new techniques.
Indeed, a variety of innovative techniques, such as microfluidics device-based isolation (reviewed in [52]), ion-exchange chromatography [60], acoustics purification [61], asymmetrical flow field-flow fractionation (AF4) [62], novel immunoisolation [63] and isolation based on lipid affinity [64], has been applied on EV/exosome isolation. Although some of the methods may be not widely applied yet, the strategy to develop and combine different techniques will offer an opportunity for improving the isolation of EVs/exosomes.
1.1.5 Characterisation of EVs/exosomes
The characterisation of EVs/exosomes is encouraged to be performed by using multiple complementary techniques to determine different properties of EVs/exosomes, such as morphology, size, density and specific protein compositions. To visualise the morphology of vesicles, various microscopy techniques can be applied, including transmission electron microscopy (TEM), cryo-electron microscopy (cryo-EM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) (reviewed in [53]). In addition to providing high-resolution morphology, TEM and SEM can be combined with immunogold labelling to detect specific surface proteins such as CD63 [47], while SEM is able to provide three-dimensional surface topology information [65, 66]. In comparison to TEM and SEM, which require extensive sample preparation before visualisation, cryo-EM enables the chemical fixation of the
samples to be avoided and thereby provides the opportunity to retain the natural state of vesicles. Through application of cryo-EM, a recent study unravelled a vast morphological diversity of an EV subpopulation sharing the same density [10]. Similarly, AFM enables direct visualisation without sample processing and both the surface topology and the local stiffness and adhesion properties of vesicles can be determined [66]. Despite the morphological information, high-resolution microscopy-based methods can be used to determine the size of vesicles, but in a less statistical manner. Dynamic light scattering (DLS), nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS) are methods that enable the quantification of vesicle size when in suspension. DLS measures bulk scattered light from vesicles undergoing continuous Brownian motion and determines the size of vesicles based on the scattered intensity [67]. NTA tracks individual vesicle scattering over time to collect information on particle velocity and diffusivity for the calculation of the vesicle size distribution [47, 68]. TRPS detects transient changes in the ionic current, generated by the transport of the vesicles through a size-tunable nanopore, as a result of which the size of vesicles is indicated [68]. Moreover, both NTA and TRPS are able to quantify the concentration of particles for calculation of the total particle number. It has been suggested that the ratio of different quantification methods (i.e. protein amount: particle number) is able partially to indicate the purity of vesicle samples [69]. The density of vesicles is often determined by sucrose or iodixanol gradient centrifugation.
Selected proteins are used as EVs/exosomes markers, which can be detected by antibody-based methods such as flow cytometry and Western blot. It is recommended to select proteins covering different categories of markers, including (i) transmembrane and/or endosomal proteins indicating the lipid- bilayer structure of EVs, such as tetraspanin CD63; (ii) cytosolic proteins, which are able to bind to membrane or to cytosolic sequences of transmembrane proteins, indicating the enclosure of intracellular materials, such as TSG101 and flotillin; (iii) proteins specific for other intracellular compartments, i.e. ER-specific protein glucose-regulated protein 94 (Grp94) [70], which are not enriched in exosomes, serving as negative markers to indicate the specificity of EV subtypes. To detect these specific proteins, Western blot is one of the most commonly used methods. Alternatively, flow cytometry can be applied to determine the surface phenotypic features of EVs in a high throughput manner [71]. Due to the limited sensitivity and resolution of conventional flow cytometry, EVs need to be bound with micrometre-sized
latex beads conjugated with antibodies such as CD63, CD81 and CD9 before detection. The pitfall of an approach like this is the lack of phenotypic signature of a single vesicle, as one bead may bind to multiple vesicles.
Recently, advanced flow cytometry with increased resolution has been developed to enable single vesicle detection [72]. In addition to protein compositions, the determination of lipid content and refractive index is suggested for characterisation of EVs [73].
1.1.6 EV-/exosome-mediated cell-to-cell communication
Upon release, EVs/exosomes travel in the extracellular space and are able to target the adjacent cells, as well as distant cells. As the first evidence showing that exosomal mRNA can be transferred and translated in the recipient cells, the secretion of EVs/exosomes was suggested as a new route to mediate cell- to-cell communication [6]. To date, a growing body of studies has intensively investigated the intercellular communication mediated by EVs/exosomes and the subsequent consequence on the regulation of recipient cells. However, the specificity of targeting EVs/exosomes to particular recipient cells and the underlying mechanisms remain largely unknown. A previous study showed that PM-derived EVs from platelets transferred tissue factor to monocytes but not to neutrophils, indicating the preferred interaction with monocytes [74].
Whereas another two studies showed that PM-derived EVs from platelets and neutrophils resulted in different effects on the same recipient cells, macrophages [75, 76]. These observations indicate that the selection of targeting cells may depend on the cell origin of EVs, the subtypes of EVs, as well as the features of recipient cells, which together contribute to the complexity of routes for EVs to interact with cells.
A variety of routes have been suggested for EVs to interact with cells, including receptor/ligand binding, membrane fusion, internalisation via (i) clathrin-, or caveolin-mediated endocytosis, (ii) phagocytosis, and (iii) micropinocytosis (reviewed in [77]). Receptor/ligand binding may trigger the direct activation of specific signalling pathways in the recipient cells, resulting in rapid responses on regulation of recipient cells. Such hypothesis is partially supported by the discovery that exosomes carry tumour necrosis factor receptor 1 (TNFR1), which is capable of binding to tumour necrosis factor-a (TNF-a) [78]. Moreover, another study showed that the blocking of exosome internalisation by cytochalasin D did not inhibit the exosome-mediated secretion of interleukin-1b (IL-1b), whereas the blocking of surface
interaction mediated by exosomal fibronectin abrogated IL-1b production, together indicating that internalisation was not essential for these biological effects mediated by exosomes [79]. However, such activation mode may depend on the functional features of the receptor and/or ligand, as well as the quantity of receptor or ligand harbouring on the surface of EVs/exosomes.
In addition to direct activation, the other consequence of receptor/ligand binding is to dock EVs/exosomes on the PM of recipient cells and facilitate the subsequent membrane fusion or internalisation. Several transmembrane proteins such as tetraspanins and integrins have been shown to play important roles in the process of cellular uptake of exosomes. A previous study demonstrated that dendritic cells (DC) internalised exosomes and, moreover, the targeting of exosomes to DCs was mediated via milk fat globule epidermal growth factor 8 (MFG-E8), CD11a, CD54, PS and the tetraspanins CD9 and CD81 on the exosome, and αv/β3 integrin, and CD11a and CD54 on the DCs [80]. Fusion with the PM of recipient cells results in the direct release of the intravesical contents. The observation of membrane fusion was achieved by using a self-quenched fluorescent lipid probe R18 [46, 81] and the fusion efficiency was enhanced by low pH condition [81]. The endocytosis-mediated uptake also requires the fusion of EV/exosome membrane with the membrane of intracellular compartments, such as endocytic compartments, to enable the intraluminal compositions of EVs/exosomes to gain access to the cytosol of recipient cells. Although the detailed intracellular fate of EVs contents is not yet fully understood, various studies have shown biological effects induced by the exosomal contents, which indirectly confirm the intracellular release of EVs/exosomes contents. For example, exosomal mRNA was translated in the recipient cells [6] and exosomal microRNA regulated the target gene expression in the recipient cells [46, 82]. Furthermore, a recent study showed that both epidermal growth factor receptor (EGFR) and androgen receptor (AR) can be transferred via EVs to the nucleus of recipient cells and the transported exogenous EGFR and AR were active and able to stimulate the nuclear pathways, respectively [83].
Due to the complexity of the in vivo environment and the fact that almost all cell types secrete EVs/exosomes which are detected in blood and other various biological fluids, it is difficult to determine the fates of EVs/exosomes in vivo.
Nevertheless, it is crucial to evaluate the biological effects of EVs/exosomes in vivo for their further application. Although EVs/exosomes have been injected into animals, due to technical limitations, the direct and accurate
tracking of the cellular internalisation and organ distribution of EVs/exosomes in vivo has been difficult to achieve. Some efforts have been made to tackle this challenge. For example, melanoma cells transfected with plasmid- expressing fusion protein consisting of luciferase and MFG-E8 were used to produce exosomes with luciferase activity [84]. These exosomes were injected intravenously and detected in the blood circulation four hours later, after which they were distributed to the liver and lungs. Another study utilised the Cre- LoxP system to induce a colour switch in recipient cells that expressed reporters when they internalised EVs released from cells that expressed Cre recombinase and, as a result, the release and internalisation of EVs in vivo were visualised [85].
1.2 Mesenchymal stem cells (MSCs)
1.2.1 Historical background of MSCs
The discovery of MSCs began in the 1960s when Friedenstein showed for the first time that the transplantation of bone marrow fragments or bone marrow cell suspension formed new bone and new bone formation required a certain density of bone marrow cells [86]. These results indicated an osteogenic potential in bone marrow, which may be driven by a subpopulation of bone marrow cells. It was subsequently determined that the bone marrow cell suspension included two main populations of cells: hematopoietic stem cells (HSCs) and non-hematopoietic stromal cells [87]. The non-hematopoietic stromal cells were initially thought only to provide physical and functional support to hematopoiesis [88], but in vitro culture showed the density- independent differences in comparison to HSCs [87]. These stromal cells exhibited capacities to adhere to plastic and initiate clonal growth in a density- insensitive fashion (a colony-forming unit-fibroblastic feature), and thus distinguished from HSCs. These features of stromal cells therefore supported the hypothesis that the bone formation in vivo upon transplantation was due to the differentiation of stromal cells, indicating the osteogenic potential of bone marrow stromal cells [89]. Afterwards, the multipotency of bone marrow stromal cells to differentiate to various mesenchymal lineages, such as osteogenic, chondrogenic, adipogenic and myogenic lineages, has been documented (reviewed in [90]). The term “mesenchymal stem cells” (MSCs) was proposed for this heterogeneous population of stromal cells with self- renewal and multipotent capacities [91]. Although there has been a large-scale
